Method of manufacturing a test article for the determination of protein

ABSTRACT

A method of manufacturing a test device for determining the presence and concentration of proteins, such as albumin or Bence Jones proteins, in a test sample. The test device includes a carrier matrix incorporating a reactant system capable of interacting with proteins to produce a visually or instrumentally detectable and/or measurable response. The carrier matrix of the device can include commonly used bibulous matrices, such as filter paper, or a nonbibulous protein-permeable strip, membrane or layer of a polymerized urethane-containing composition. In addition, a reactant system, including a dual indicator reagent system, such as bromophenol blue, methyl orange and, if necessary, a suitable buffer, is incorporated into the carrier matrix to provide improved color resolution and increased sensitivity to proteins, thereby affording a more accurate and trustworthy protein assay of test samples, such as urine. Furthermore, by incorporating the dual indicator reagent system into a carrier matrix including a polymerized urethane-containing composition, a dry phase assay for low molecular weight proteins, such as Bence Jones proteins, can be performed on a liquid test sample.

RELATED APPLICATIONS

This is a division of application Ser. No. 620,083, filed on Nov. 30,1990, now U.S. Pat. No. 5,077,222; which is a division of applicationSer. No. 251,297, filed on Sep. 30, 1988, which is now U.S. Pat. No.5,049,358.

FIELD OF THE INVENTION

The present invention relates to a device and a method of assaying atest sample for the presence and concentration of proteins. Moreparticularly, the present invention relates to a new and improved methodand device for assaying a liquid, such as urine, for proteins byutilizing a device having a dual indicator reagent composition as thereactant composition. The dual indicator reagent composition isincorporated into a carrier matrix, such that a detectable and/ormeasurable response occurs upon contact of the dual indicator reagentcomposition with a protein-containing test sample. The dual indicatorreagent composition provides improved color resolution and increasedprotein sensitivity in order to more accurately detect and/or measure,either visually or by instrument, the protein content of a liquid testsample. In addition, the present invention relates to using a dualindicator reagent composition, incorporated into a carrier matrixcomprising a protein-permeable strip, membrane or layer of a polymerizedurethane-containing composition, in a method to determine the presenceand/or concentration of low molecular weight proteins, like Bence Jonesproteins, in a test sample by a dry phase, test strip assay procedure.

BACKGROUND OF THE INVENTION AND PRIOR ART

Albumin is the most abundant plasma protein, generally constitutingslightly over one-half of the total protein in mammalian plasma. In thehuman body, albumin has the important role of regulating the waterbalance between blood and tissues, and of functioning as a transportmolecule for various compounds, such as bilirubin, fatty acids,cortisol, thyroxine and drugs such as sulfonamides and barbiturates,that are only slightly soluble in water. An albumin deficiency canrestrict the transport of slightly water soluble materials throughoutthe body and a deficiency is signaled in an individual by an abnormalaccumulation of serous fluid, or edema. Therefore, it is clinicallyimportant to determine whether an individual has a deficiency of serumalbumin.

Likewise, it is clinically important to determine if an individual isexcreting an excess amount of protein. A normal functioning kidney formurine in an essentially two step process. Blood flows through theglomerulus, or glomerular region of the kidney. The capillary walls ofthe glomerulus are highly permeable to water and low molecular weightcomponents of the blood plasma. Albumin and other high molecular weightproteins cannot pass through these capillary walls and are essentiallyfiltered out of the urine so that the protein is available for use bythe body. The liquid containing the low molecular weight componentspasses into the tubules, or tubular region, of the kidney where thereabsorption of some urine components, such as low molecular weightproteins; secretion of other urine components; and concentration of theurine occurs. As a result, through the combined processes of theglomerulus and tubules, the concentration of proteins in urine should beminimal to non-existent. Therefore, abnormally high amounts of albuminand/or low-molecular weight proteins in urine must be detected andrelated to a physiological dysfunction.

The relatively high concentration of albumin in the urine of anindividual usually is indicative of a diseased condition. For example,the average normal concentration of protein in urine varies from about 2mg/dL to about 8 mg/dL, with approximately one-third of the totalurinary protein being serum albumin. However, in a majority of diseasedstates, urinary protein levels increase appreciably, such that albuminaccounts for from about 60 percent to about 90 percent of the excretedprotein. The presence of an abnormal increased amount of protein in theurine, known as proteinuria, is one of the most significant indicatorsof renal disease, and may be indicative of various other non-renalrelated diseases.

Therefore, in order to determine if an individual has an albumindeficiency and/or to determine if an individual excretes an excessamount of protein, and in order to monitor the course of medicaltreatment to determine the effectiveness of the treatment, simple,accurate and inexpensive protein detection assays have been developed.Furthermore, of the several different assay methods developed for thedetection and/or measurement of protein in urine and serum, the methodsbased on dye binding techniques have proven especially useful becausedye binding methods are readily automated and provide reproducible andaccurate results.

In general, dye binding techniques utilize pH indicator dyes that arecapable of interacting with a protein, such as albumin, and that arecapable of changing color upon interaction with a protein absent anychange in pH. When a pH indicator dye interacts with, or binds to, aprotein, the apparent pK_(a) (acid dissociation constant) of theindicator dye is altered and the dye undergoes a color transition,producing the so-called "protein-error" phenomenon. In methods utilizingthe dye binding technique, an appropriate buffer maintains the pHindicator dye at a constant pH to prevent a color transition of the pHindicator dye due to a substantial shift in pH. Due to the"protein-error" phenomena, upon interaction with the protein, the pHindicator dye undergoes a color transition that is identical to thecolor change arising because of a change in the pH. Examples of pHindicator dyes used in the dry phase assay of proteins that are capableof interacting with or binding to proteins and exhibiting"protein-error" color transitions include tetrabromophenol blue andtetrachlorophenol-3,4,5,6-tetrabromosulfophthalein.

Although pH indicator dyes have been used extensively in protein assays,several problems and disadvantages still exist in protein assay methodsutilizing indicator dyes. For example, methods based upon pH indicatordyes either cannot detect or cannot quantitatively differentiate betweenprotein concentrations below approximately 15 mg/dL. In addition,although several simple semiquantitative tests and several complexquantitative tests are available for the determination of the totalprotein content in a test sample, the majority of these assay methods,with the notable exception of the simple colorimetric reagent teststrip, require the precipitation of protein to make quantitative proteindeterminations.

The colorimetric reagent test strip utilizes the previously discussedability of proteins to interact with certain acid-base indicators and tolater the color of the indicator without any change in the pH. Forexample, when the indicator tetrabromophenol blue is buffered tomaintain a constant pH of approximately 3, the indicator imparts ayellow color to solutions that do not contain protein. However, forsolutions containing protein, the presence of protein causes thebuffered dye to impart either a green color or a blue color to solution,depending upon the concentration of protein in the solution.

Some colorimetric test strips used in protein assays have a single testarea consisting of a small square pad of a carrier matrix impregnatedwith a buffered pH indicator dye, such as tetrabromophenol blue. Othercolorimetric test strips are multideterminant reagent strips thatinclude one test area for protein assay as described above, and furtherinclude several additional test areas on the same strip to permit thesimultaneous assay of other urinary constituents. For both types ofcolorimetric test strips, the assay for protein in urine is performedsimply by dipping the colorimetric test strip into a well mixed,uncentrifuged urine sample, then comparing the resulting color of thetest area of the test strip to a standardized color chart provided onthe colorimetric test strip bottle.

For test strips utilizing tetrabromophenol blue, buffered at pH 3, asthe indicator dye, semiquantitative assays for protein can be performedand are reported as negative, trace, or one "plus" to four "plus". Anegative reading, or yellow color, indicates that the urine contains noprotein, as demonstrated by the lack of a color transition of theindicator dye. A trace reading may indicate from about 5 to about 20mg/dL of protein in the urine. The one "plus" to four "plus" readings,signified by color transitions of green through increasingly dark shadesof blue, are approximately equivalent to urine protein concentrations of30, 100, 300, and over 2000 mg/dL, respectively, and serve as reliableindicators of increasingly severe proteinuria.

In accordance with the above-described method, an individual can readilydetermine, visually, that the protein content of a urine sample is inthe range of 0 mg/dL to about 30 mg/dL. However, the colordifferentiation afforded by the presently available commercial teststrips is insufficient to allow an accurate determination of proteincontent in urine between 0 mg/dL and about 15 mg/dL. The inability todetect and differentiate between low protein concentrations is importantclinically because a healthy person usually has a urine protein level inthe range of about 10 mg/dL to about 20 mg/dL. Therefore, it could beclinically important to know more precisely the urine protein content ofan individual, rather than merely estimating the protein content at somevalue less than about 30 mg/dL.

Of course, the protein content of a urine sample can be determined moreprecisely by semiquantitative protein precipitation techniques or byquantitative 24 hour protein precipitation techniques. However, thesetests are time consuming and relatively expensive. Furthermore, theprecipitation tests must be run in a laboratory by trained personnel,and therefore are unavailable for the patient to perform at home toquickly determine urine protein content and to monitor the success orfailure of a particular medical treatment.

Therefore, it would be extremely advantageous to have a simple, accurateand trustworthy method of assaying urine for protein content that allowsvisual differentiation of protein levels in the ranges of 0 mg/dL toabout 10 mg/dL, about 10 mg/dL to about 20 mg/dL, and about 20 mg/dL toabout 30 mg/dL, and upwards to between about 100 mg/dL to about 300mg/dL. By providing such an accurate method of determining urine proteinconcentration in an easy to use form, such as a dip-and-read test strip,the urine assay can be performed by laboratory personnel to affordimmediate test results, such that a diagnosis can be made without havingto wait up to one day for assay results and medical treatment can becommenced immediately. In addition, the test strip method can beperformed by the patient at home to more precisely monitor low levels orprotein in urine and/or the success of the medical treatment the patientis undergoing.

As will be described more fully hereinafter, the method of the presentinvention allows the fast, accurate and trustworthy protein assay ofurine by utilizing a test strip that includes a dual indicator reagentcomposition. The dual indicator reagent composition improves the visualcolor resolution, and therefore the sensitivity, of the assay, therebyallowing urine protein concentrations to be accurately determined atlevels of approximately 30 mg/dL or less. In addition, the method of thepresent invention can be used to determine the presence and/orconcentration of low molecular weight proteins, such as Bence Jonesproteins, in a test sample. All prior art assay techniques for lowmolecular weight proteins involve immunoelectrophoresis method of heatest methods that are time consuming, relatively expensive and are notamenable for use by the patient at home to detect low molecular weightproteins in urine.

Bence Jones proteins belong to a class of urinary proteins having a lowmolecular weight of approximately 20,000 mg/dL and that are small enoughto pass through the glomerular filters of the kidney. However, the BenceJones proteins usually are reabsorbed in the tubular section of thekidney. Therefore, the concentration of Bence Jones proteins isnegligible in the urine of a healthy person. As a result, a significantamount of Bence Jones proteins in urine generally is clinicallysignificant. Overall, the detection and measurement of the concentrationof low molecular weight proteins in urine is important because certaindiseases are characterized by the excretion of specific low molecularweight proteins (globulins) rather than by diffuse proteinuriacharacterized by elevated albumin levels.

For example, the Bence Jones proteins represent a portion of the highmolecular weight plasma myeloma globulin, and therefore are found inincreased amounts in the urine of more than one-half of the patientssuffering from multiple myeloma or leukemia. Bence Jones proteinuriaalso is found in the urine of many patients suffering frommacroglobulinemia and primary systemic amyloidosis. In addition, anincreased excretion of a specific globulin that is similar to BenceJones proteins occurs in Franklin's disease; and patients with renaltubular disorders, such as the Fanconi syndrome, show a substantialincrease in the quantity of globulins excreted in the urine.Accordingly, investigators have searched for a simple assay for lowmolecular weight proteins because the dye-binding method used incommercially available test strips is insensitive to low molecularweight proteins, like Bence Jones proteins. Surprisingly andunexpectedly, the method of the present invention provides a techniqueto detect and measure the concentration of low molecular weightproteins, like Bence Jones proteins using a dual indicator reagentcomposition incorporated into a polymerized urethane-containing film,layer or membrane having an appropriate pore size.

The Bence Jones proteins differ from all other urinary proteins in thatthey coagulate upon heating to temperatures between about 45° C. andabout 60° C., and then redissolve on further heating to the boilingpoint of test sample. This peculiar characteristic of Bence Jonesproteins has been the basis of all qualitative and semiquantitativedeterminations for Bence Jones proteins. The dye binding technique usedin commercially available test strips has proved insensitive to BenceJones proteins because the much greater relative concentration of highermolecular weight proteins, such as albumin, in the urine of a healthyindividual effectively interferes with and masks the presence of BenceJones proteins. Furthermore, it is inconvenient and costly to separatethe albumin from Bence Jones proteins, thereby negating the utility ofseparating the albumin from the Bence Jones proteins before using a dryphase test strip.

As a result, dry phase test strips are presently unavailable to test forthe presence and concentration of Bence Jones proteins in urine.However, incorporating the highly sensitive dual indicator reagentcomposition of the present invention into a carrier matrix having asufficiently small pore size prevents the albumin content of the urinesample from penetrating the carrier matrix and interacting with the dualindicator reagent composition to cause a color transition. However, thecarrier matrix is of sufficient pore size to allow Bence Jones proteinsto penetrate the carrier matrix and to interact with the dual indicatorreagent composition to cause a color transition.

Proteinuria resulting either from abnormally high albumin levels or thepresence of low-molecular weight proteins depends upon the precisenature of the clinical and pathological disorder and upon the severityof the specific disease. Proteinuria can be intermittent or continuous,with transient, intermittent proteinuria usually being caused byphysiologic or functional conditions rather than by renal disorders.Therefore, accurate and thorough assays or urine and other test samplesfor protein must be available for both laboratory and home use. Theassays must permit the detection and measurement of the proteins ofinterest, either albumin and/or Bence Jones proteins, such that acorrect diagnosis can be made and correct medical treatment implemented,monitored and maintained. In addition, it would be advantageous if theprotein assay method, both for high molecular weight proteins, likealbumin, and low molecular weight proteins, like Bence Jones proteins,could be utilized in a dip-and-read format for the easy and economical,qualitative and/or semiquantitative determination or protein in urine orother test samples.

Furthermore, any method of assaying for protein in urine or other testsamples must yield accurate, trustworthy and reproducible results byutilizing a composition that undergoes a color transition as a result ofan interaction with protein, and not as a result of a competing chemicalor physical interaction, such as a pH change or preferential interactionwith a test sample component other than protein. Moreover, it would beadvantageous if the protein assay method is suitable for use both in wetassays and in dry reagent strips for the rapid, economical and accuratedetermination of protein in urine or other test samples. Additionally,the method and composition utilized in the assay for protein should notadversely affect or interfere with the other test reagent pads that arepresent on multiple test pad strips.

Prior to the present invention, no known method of assaying urine orother test samples for proteins included a dual indicator reagentcomposition that improves color resolution of the assay and increasesthe sensitivity of the assay at lower protein concentration levels, suchthat accurate and trustworthy protein assays can be made for proteinconcentrations of about 30 mg/dL and below. In addition, although a dryphase chemistry test strip utilizing a single dye, such astetrabromophenol blue ortetrachlorophenol-3,4,5,6-tetrabromosulfonephthalein, has been usedextensively for several years, no dry phase test strip has incorporatedtwo dyes to improve visual color resolution, and therefore increasesensitivity, at lower protein concentration levels. Furthermore, untilthe method of the present invention, dry phase test strip procedureswere available principally to test for total protein concentration,i.e., for albumin. However, surprisingly and unexpectedly, the method ofthe present invention permits the dry phase test strip assay or urineand other test samples for low molecular weight proteins, such as BenceJones proteins.

The prior art contains numerous references on the wet phase and the dryphase chemistry utilized in the pH indicator dye method of assayingurine for proteins. For example, Keston U.S. Pat. No. 3,485,587discloses the basic dye binding technique used to assay for proteins ata constant pH. Keston teaches utilizing a single indicator dye,maintained at a constant pH slightly below the pK_(a) (acid dissociationconstant) of the dye, to determine the presence and/or concentration ofalbumin by monitoring the color transition of the dye.

In contrast to the prior art, and in contrast to the presently availablecommercial test strips, the method of the present invention providesincreased sensitivity in the detection and measurement of proteins inurine by utilizing a combination of indicator dyes, such that accurateprotein levels of about 30 mg/dL and below can be determined.Unexpectedly and surprisingly, the method of the present invention alsoallows the simple and essentially immediate detection and measurement oflow levels of Bence Jones proteins; a method heretofore impossiblebecause of interference by the relatively high concentration of abluminin the urine sample. Hence, in accordance with the method of the presentinvention, new and unexpected results are achieved in the dry phasereagent strip assay, and the wet assay, of urine and other test samplesfor proteins, including low molecular weight proteins, by utilizing adual indicator reagent composition incorporated into a carrier matrixhaving an appropriate pore size.

SUMMARY OF THE INVENTION

In brief, the present invention is directed to a new and improved testdevice, method of manufacturing the test device, and method ofdetermining the presence and/or concentration of a component in a testsample. The device includes a carrier matrix incorporating a reactantcomposition capable of interacting with a test sample component toproduce a detectable response. For home use, the reactant compositionproduces a visually detectable response. For laboratory use, thereactant composition produces a response that is detectable visually orby instrument. The carrier matrix of the device of the present inventioncomprises such bibulous porous materials as filter paper, or a new andimproved nonbibulous protein permeable strip, layer or membrane of apolymerizable urethane-containing material. A reactant composition canbe homogeneously incorporated into the polymerizable carrier matrixprior to or after complete curing of the matrix, and the carrier matrixthen holds the reactant composition homogeneously throughout the carriermatrix in a known concentration while maintaining carrier matrixpenetrability of the predetermined component after complete curing ofthe carrier matrix.

More particularly, the present invention is directed to a method ofassaying urine or other test samples for proteins by utilizing a new andimproved dual indicator reagent composition. It has been demonstratedthat employing a combination of indicator dyes, capable of undergoingcolor transitions in approximately the same pH range, affords improvedcolor resolution and increased sensitivity at low protein concentrationranges. In accordance with an important feature of the presentinvention, the qualitative and/or semiquantitative determination ofprotein levels between 0 mg/dL and about 2000 mg/dL, and especiallybetween 0 mg/dL and about 30 mg/dL, in urine and other test samples isaccomplished. By utilizing the dual indicator reagent composition of thepresent invention in clinical test methods, the qualitative and/orsemiquantitative concentration of proteins, such as albumin, in urine orother test sample can be more accurately determined because improvedcolor resolution afforded by the combination of dyes increases thesensitivity of the method to low concentrations of protein. Furthermore,surprisingly and unexpectedly, the dual indicator reagent compositionincorporated into a test device including a new and improvedpolyurethane-based carrier matrix allows the detection and measurementof low molecular weight proteins, such as Bence Jones proteins, in urineand other test samples.

Therefore, it is an object of the present invention to provide a new andimproved method and test device for determining the relativeconcentration of a chemical compound in a liquid.

Another object of the present invention is to provide a simple,trustworthy, accurate and reproducible method of assaying urine or othertest samples for proteins.

Another object of the present invention is to provide a new and improvedprotein interactive test device for interaction with protein in a testfluid to produce a visible change, such as a change in color, of thetest device, indicative of the protein concentration in the test fluid.

Another object of the present invention to provide a method of assayingurine or other liquid test samples for albumin or low molecular weightproteins, such as Bence Jones proteins.

Another object of the present invention is to provide a method ofassaying urine or other liquid test samples that provides improvedvisual color resolution and increased sensitivity to low proteinconcentrations.

Yet another object of the present invention is to provide a method ofassaying urine or other liquid test samples that is sensitive to proteinconcentrations of less than about 15 mg/dL and that semiquantitativelydiscriminates between protein levels of from 0 mg/dL to about 2000mg/dL, and especially from 0 mg/dL to about 30 mg/dL.

Another object of the present invention is to provide a method ofassaying urine or other test liquids that utilizes a dual indicatorreagent composition.

Another object of the present invention is to provide a method ofassaying urine or other test liquids by utilizing a dual indicatorreagent composition that, when buffered in the pH range slightly belowthe color transition pH of the indicator components of the composition,can interact with proteins and undergo a detectable and measurable colortransition to establish the presence and concentration of protein in thetest sample.

Another object of the present invention is to provide a dual indicatorreagent composition that, when appropriately buffered, can interact withproteins and undergo a visually and/or instrumentally differentiablecolor transition to allow the semiquantitative determination of theconcentration of protein in the urine or other liquid samples at levelsbetween 0 mg/dL and about 2000 mg/dL, and especially between 0 mg/dL andabout 30 mg/dL.

Another object of the present invention is to provide a method ofassaying urine or other test samples for the presence and concentrationof low molecular weight proteins.

Still another object of the present invention is to provide a method ofassaying a liquid sample for low molecular weight proteins, includingBence Jones proteins, by utilizing a dual indicator reagent composition.

Another object of the present invention is to provide a method ofassaying for Bence Jones proteins by incorporating the dual indicatorreagent composition into a dry phase detection device, comprising acarrier matrix having a porosity sufficient to allow penetration by lowmolecular weight proteins, such as Bence Jones proteins, but to precludepenetration by higher molecular weight proteins, such as albumin.

Another object of the present invention is to provide a method ofmanufacturing a detection device for low molecular weight proteinscomprising a dual indicator reagent composition incorporated into acarrier matrix of suitable porosity.

Another object of the present invention is to provide a new and improvedtest device and method of manufacturing the test device including acarrier matrix having incorporated therein during manufacture thereof, areactant composition capable of interacting with a chemical compound ina test sample, wherein the carrier matrix comprises a polymerizableurethane-containing composition.

Another object of the present invention is to provide a reagent stripcomprising a carrier matrix comprising a polymerizableurethane-containing composition capable of relatively homogeneousmixture with a dual indicator reactant composition prior to during andpermeable to low molecular weight proteins after curing.

Another object of the present invention is to provide a new and improvedtest device and method of manufacturing the test device for sensing thepresence of a chemical compound in a liquid, where the chemical compoundis capable of permeating a polymer-based carrier matrix and capable ofreacting with a dual indicator reagent composition incorporated into thecarrier matrix during manufacture prior to complete curing of thecarrier matrix or after complete curing of the carrier matrix.

A still further object of the present invention is to provide a new andimproved dry phase test strip capable of incorporating a dual indicatorreactant composition into the carrier matrix during or after manufactureto achieve a test strip of new and unexpected precision in proteinresponse.

Another object of the present invention is to provide a new and improvedreagent test strip, capable of interacting with a predetermined proteincomponent in an assay medium, having a dual indicator reactantcomposition incorporated into a carrier matrix comprising a curedpolymer layer, film or membrane permeable to the predetermined proteincomponent of the assay medium.

Another object of the present invention is to provide a new and improvedtest device for the quantitative analysis of proteins, including lowmolecular weight proteins.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and advantages and novel features of thepresent invention will become apparent from the following detaileddescription of the preferred embodiments of the invention illustrated inthe accompanying figures illustrating the enhanced color resolution ofthe color transition in the reagent test strips and the increasedsensitivity to proteins, permitting more accurate semiquantitativeanalyte determinations:

FIG. 1 is a color space plot showing the assay of liquid samplescontaining 0, 10, 50 and 100 mg/dL of albumin respectively and 100 mg/dLof Bence Jones proteins using a dry phase test strip comprising a filterpaper bibulous matrix incorporating the single indicator dyetetrabromophenol blue (TBPB);

FIG. 2 is a color space plot showing the assay of liquid samplescontaining 0, 10, 50 and 100 mg/dL of albumin respectively and 100 mg/dLof Bence Jones proteins using a dry phase test strip comprising a filterpaper bibulous matrix incorporating dual indicator dyes,tetrabromophenol blue (TBPB) and methyl orange (MO);

FIG. 3 is a color space plot showing the assay of liquid samplescontaining 0, 10, 50 and 100 mg/dL of albumin respectively and 100 mg/dLof Bence Jones proteins using a dry phase test strip comprising acarrier matrix comprising a polymerized urethane-containing filmincorporating a single indicator dye, tetrabromophenol blue (TBPB); and

FIG. 4 is a color space plot showing the assay of liquid samplescontaining 0, 10, 50 and 100 mg/dL of albumin respectively and 100 mg/dLof Bence Jones proteins using a dry phase test strip comprising acarrier matrix comprising a polymerized urethane-containing filmincorporating dual indicator dyes, tetrabromophenol blue (TBPB) andmethyl orange (MO).

DETAILED DESCRIPTION OF THE INVENTION

In accordance with the method of the present invention, the qualitativeand/or semiquantitative assay for proteins, including albumin and/or lowmolecular weight proteins, in urine and other test samples isaccomplished by utilizing a dual indicator reagent composition. Byemploying a combination of suitable indicator dyes, visual colorresolution is improved over assays employing a single indicator dye, andthe sensitivity of the assay to low concentration levels of protein isincreased. The improved color resolution and increased sensitivity tolow protein levels afforded by the method of the present invention isespecially useful in urine assays.

Present-day commercial assays are incapable of differentiating betweenprotein levels ranging from 0 mg/dL to about 30 mg/dL, and especiallyfrom 0 mg/dL to about 15 mg/dL. Differentiating between low proteinconcentration levels is clinically important in the art because a rangeof from about 10 mg/dL to about 20 mg/dL is used as the normal urineprotein level for a healthy individual, therefore urine protein levelsfrom 0 mg/dL to about 10 mg/dL may indicate a potential proteindeficiency that can cause physiological imbalances and urine proteinlevels greater than about 20 mg/dL may indicate an excessive excretionof proteins that can signify a diseased state. It should be noted thatin regard to urine protein concentrations in the relatively high range,such as from about 100 mg/dL to about 2000 mg/dL, the method of thepresent invention still affords improved color resolution and increasedsensitivity to urine protein concentration, however such clinicalbenefits are less critical in this concentration range since such highprotein levels are definitely indicative of an abnormal physiologicalstate that must be investigated further.

In further regard to urine assays, the presence of low levels of lowmolecular weight proteins, such as Bence Jones proteins, is indicativeof specific diseased states, such as leukemia or multiple myeloma.Therefore, in accordance with another important feature of the deviceand method of the present invention, the improved color resolutionafforded by the use of the dual indicator reagent composition and theresulting increased sensitivity to low levels or protein in urineprovides a technique to detect and measure the concentration of lowmolecular weight proteins present in urine. Therefore, as will bediscussed more fully hereinafter in the detailed description of theinvention, a method and device is available to test either for totalurine protein content in urine, or for the low molecular weight proteincontent in urine by eliminating the interferences caused by the highermolecular weight proteins, such as albumin.

Furthermore, it will become apparent that in addition to assaying urine,the method and device of the present invention also can be used todetermine the presence and semiquantitive concentration of albumin inblood plasma and serums; and more generally, the albumin content of mayother albumin-containing fluids as well. In accordance with anotherimportant feature of the present invention, the method and compositionof the present invention can be employed both in aqueous, liquid phaseassays and, to achieve the full advantage of the present invention, indry phase, test pad assays to determine the presence and/orconcentration of proteins in urine or other liquid test samples.

Surprisingly and unexpectedly, it has been found that combining twosuitable indicator dyes, each having the ability to interact withproteins and undergo a detectable and measurable color transition whilemaintained at a constant pH, demonstrated improved color resolution andincreased sensitivity to low protein concentrations when used in adye-binding technique to determine the presence and/or concentration ofproteins in a test sample. The dye-binding technique using the dualindicator reagent composition provides a more accurate, trustworthy andclinically significant semiquantitative assay for protein. Presently,both liquid phase assays and commercially available dry phase, teststrip assays utilize only a single dye, such as tetrabromophenol blue ortetrachlorophenol-3,4,5,6-tetrabromosulfonephthalein, as the indicatordye to determine the presence and/or semiquantitative concentration ofprotein in a test sample.

The dyes presently used in assays for protein interact with proteins andundergo a color transition due to the protein-error phenomena whenmaintained at the proper, constant pH. The protein-error phenomena iffully described in Keston U.S. Pat. No. 3,485,587, wherein the variousdyes, the correct pH ranges and the buffers required to observe theprotein-error phenomena are disclosed. The Keston patent basicallydescribes the present day dry phase test strips employed to assay fortotal protein content in urine. These total protein test stripsgenerally include an indicator dye that normally undergoes a colortransition at a strongly acidic pH or 5 or below, and a buffer tomaintain the pH of the indicator dye slightly below the pH of colortransition for the dye. A sufficient buffering of the indicator dyeessentially assures that the dye changes color due to an interactionwith protein rather than due to a pH change occurring upon contact withthe test sample.

In accordance with an important feature of the present invention, it hasbeen demonstrated that a judicious selection of a pair of indicatordyes, properly buffered at a suitable pH, provides a more accurate andtrustworthy assay for total protein content in liquid samples.Furthermore, both suprisingly and unexpectedly, by incorporating a dualindicator reagent composition in a dry phase test stick comprising acarrier matrix comprising a polymerized urethane-containing film, layeror membrane, the selective detection and measurement of low molecularweight proteins in a test sample is accomplished. In addition, thedetection and measurement of the low molecular weight protein isachieved without having to separate the predominant, competing andinterfering higher molecular weight proteins, such as albumin, from thetest sample. Therefore, a time-consuming and expensive additionalmanipulative step is avoided. Furthermore, a method of fast, accurate,reproducible and trustworthy assays, performable at home or in thelaboratory to yield essentially immediate assay results for lowmolecular weight proteins, is achieved.

In order to achieve the benefits afforded by the present invention, itis imperative that the dual indicator reagent composition includes asuitable combination of indicator dyes. In contrast both to the priorart and to presently available commercial assays that utilize a singleindicator dye, the incorporation of two indicator dyes, each having anessentially identical color transition pH range and neither undergoingan identical color transition, improves the color resolution anddifferentiation, both visually and instrumentally, of the colortransition occurring upon interaction with proteins. Therefore, thesensitivity of the protein assay, especially at relatively low proteinconcentrations, is increased.

The method of the present invention utilizes the "protein-error"phenomena previously discussed. However, the incorporation of twoindicator dyes into the dual indicator reagent composition introducesthe principle of competitive interaction between each of the twoindicator dyes for the available protein in the test sample at acontrolled pH. As previously described, when a pH indicator dyeinteracts with a protein, the apparent pKa of the dye is altered and acolor transition occurs producing the so-called "protein-error"phenomenon. However, by employing two indicator dyes, each having anapproximately identical color transition pH range, two color transitionsare observed simultaneously. By adjusting the relative amounts of thetwo indicator dyes, in relation to the ability of each dye to interactwith protein and in relation to the actual color transition and theintensity of color transition of each dye, a more spectacular colordevelopment is achieved, therefore improving color resolution anddifferentiation upon interaction with proteins and accordinglyincreasing assay sensitivity.

In general, any two pH indicator dyes can be utilized in the method ofthe present invention, provided that three basic requirements aresatisfied. Initially, it is of primary importance that each dye iscapable of interacting with proteins and undergoing a detectable andmeasurable color transition in response to the protein interaction. Theindicator dyes utilized in the dual indicator reagent composition mustpreferentially interact with proteins as opposed to any competingchemical or physical interactions with non-protein components in thetest sample. Any appreciable competing interactions with non-proteincomponents could lead to false and erroneous assays concerning thepresence and amount of protein in the test sample. For example, theproper buffering of the indicator dyes precludes the possibility of acolor transition occurring because of a pH change in all cases exceptthose wherein the test sample is sufficiently alkaline to overcome theeffect of the buffers.

In addition, it is important that each dye has a relatively similaraffinity to interact with proteins. It has been found that if one dyehas more than an approximately ten to approximately fifteen timesgreater affinity to proteins than the second dye, erroneous and falseassays may result because preferential interaction of one dye with theprotein produces a color transition that does not accurately correlateto the concentration of protein in the sample. The inability of thesecond dye to effectively interact with the proteins can lead toerroneously high or erroneously low results because only the first dyewill undergo a color transition in response to the protein interaction,and this color transition will not be balanced and modified by a secondcolor transition occurring in response to the interaction of the seconddye with the proteins present in the test sample.

Secondly, each of the indicator dyes utilized in the dual indicatorreagent composition must undergo a color transition at approximately thesame pH range. Normally, a difference in pH range for color transitionbetween the two dyes of up to about 0.5 pH units is acceptable; however,to achieve the full advantage of the present invention, the differencein pH range for color transition between the two dyes is preferablylimited to about 0.2 to about 0.3 pH units. An equal or approximatelyequal, pH color transition range is required because in the dye bindingtechnique the indicator dye is maintained at a constant pH, usuallyslightly below the color transition pH range of the dye, to assure thatthe color transition occurs because of an interaction with a protein andnot because of a pH change. In accordance with the method of the presentinvention, each dye is buffered to a pH value slightly below the pHrange wherein the dye changes color, in order for each dye to undergoits maximum color transition, and therefore most appreciably improvecolor resolution and most substantially increase assay sensitivity.Therefore, to maximize the color transition for the dual indicatorreagent composition as a whole, the two indicator dyes must undergo acolor transition at approximately the same pH range.

Finally, the dyes employed in the dual indicator reagent compositionmust undergo color transitions that do not mutually interfere with oneanother. For example, the benefits of improved color resolution andincreased assay sensitivity can be defeated or minimized if each dyeundergoes a color transition from a less intense color to a more intensecolor. Similarly, the benefits afforded by the present invention alsoare minimized or negated in situations wherein the first dye undergoes acolor transition to match the original color of the second dye, and thesecond dye undergoes a color transition to match the original color ofthe first dye. For example, if at a constant pH, and prior tointeraction with a protein, the first dye is red in color and the seconddye is colorless; then upon interaction with protein in a test sample,the first dye undergoes a color transition from red to colorless and thesecond dye undergoes a color transition from colorless to red, thebenefits of improved color resolution and assay sensitivity arediminished or negated, regardless of whether the assay is monitoredvisually or by instrument. Therefore, in order to achieve the fulladvantage of the present invention, the dyes employed in the dualindicator reagent composition are selected such that one dye changesfrom a more intense color to a less intense color, and the second dyechanges from a less intense color, that differs from the less intensecolor of the first dye, to a more intense color that differs from themore intense color of the first dye.

It has been found that any pH indicator dye can be used in the method ofthe present invention, provided that both dyes of the dual indicatorreagent composition are capable of interacting with proteins to undergoa sufficient and contrasting color transition at approximately the samepH range. Depending upon several chemical and physical parameters, suchas ability to interact with proteins, intensity of the color transitionand chemical compatibilty between the dyes, the weight ratio of thefirst indicator dye in the dual indicator reagent composition to thesecond indicator dye of the reagent composition can range fromapproximately 5 to 1 to approximately 1 to 5, and preferentially fromabout 3 to 1 to about 1 to 3. The exact ratio of the first indicator dyeto the second indicator dye of the dual indicator reagent compositioncan be determined by those skilled in the art of designing test kits inorder to produce an assay for proteins having maximum visual colorresolution and maximum sensitivity. The indicator dyes utilized in thedual indicator reagent composition of the present invention can beprepared by methods well known to persons in the art. Furthermore,several indicator dyes that are useful in the method of the presentinvention are well known acid-base indicator dyes that are presentlyavailable commercially.

A combination of indicator dyes as described above is utilized as anindicator reagent composition in an improved method to determine thepresence and/or the semiquantitative concentration of protein in urineor other liquid test samples. It has been demonstrated that the dualindicator reagent composition of the present invention interacts withproteins to produce a differentiable and measurable color transition,either visually and/or by instrument, due to the "protein-error"phenomena. However, in addition to the combination of dyes, the dualindicator reagent composition of the present invention may require asufficient amount of a proper buffer, such that the dyes will not changecolor as a result of a pH shift, but will change color upon contact andinteraction with proteins to accurately establish the presence and/orsemiquantitative concentration of protein in the test sample.

Further, it has been demonstrated that any of various known types ofbuffers can be used in the dual indicator reagent composition of thepresent invention. The function of the buffer is to maintain the reagentcomposition at a substantially constant pH to produce the desired colortransition in the indicators because of the presence of proteins and toessentially eliminate color changes due to a variation in the pH of theprotein-containing test sample. As a result, the amount of bufferincorporated into the dual indicator reagent composition depends uponthe nature of the test sample. The quantity of buffer usually fallsbetween about 100 millimolar (mM) and about 500 millimolar, although inparticular cases the amount of buffer can be above or below this range.The nature of the buffer used will depend upon, and vary with, theindicators incorporated into the dual indicator reagent composition.However, it has been found that for optimum results, the pH of thereagent composition generally should be maintained at a pH value onlyslightly below the pH range wherein the two indicator dyes of thereagent composition undergo a color transition. A method of determininga suitable buffered pH value for the particular indicator dyes of thereagent composition and of determining the particular buffer than can beused in the dual indicator reagent composition is found in Keston, U.S.Pat. No. 3,485,587.

Although the use of a buffer in the present dual indicator reagentcomposition is preferred, a buffer is not essential in all cases. Forexample, in special cases it may be desirable to add a buffer to theurine or other test sample before the test sample contacts the dualindicator reagent composition. Also the test sample may already containa buffer of the proper type and in the proper amount to maintain thecomposition at a constant pH, or the dual indicator dye composition maybe insensitive to pH changes. In such cases, the two indicator dyes canbe the sole active ingredients in the dual indicator reagentcomposition. However, it should be understood that optional ingredients,such as surfactants, that do not materially alter the nature and thefunction of the indicator dyes and/or the buffer and that do notinterfere with the protein assay, also can be included in the dualindicator reagent composition. Likewise, other such non-essentialingredients include nonactive background dyes, polymers andplasticizers.

Upon contact with the urine or other test sample, a color transition ofthe dual indicator reagent composition demonstrates the presence ofprotein. Furthermore, the intensity and degree of the color transitioncan be used to determine the semiquantitative concentration of proteinin the test sample by comparing or correlating the color produced by thetest sample to color, produced by solutions having a known concentrationof protein. In accordance with an important feature of the presentinvention, it has been demonstrated that the dual indicator reagentcomposition provides a sufficiently resolved and differentiated colortransition such that the amount of protein in the test sample can bemeasured and accurately determined without the use of color-measuringinstruments, such as spectrophotometers or colorimeters. However, ifdesired, such color-measuring instruments can be used to measure thedifference in color degree and intensity between the test sample and asolution of known albumin concentration.

Accordingly, an assay for protein that utilizes a suitably buffered dualindicator reagent composition improves the accuracy and reliability ofthe assay and also increases physician confidence in the assay.Additionally, because of the number of urine assays for protein beingperformed at home by the untrained patient, as opposed to trainedphysicians or technicians in the laboratory, it is imperative to provideaccurate and reliable semiquantitative assay methods for protein contentin the urine.

In accordance with an important feature of the present invention, TABLEI tabulates representative pH indicator dyes that can be used as proteinindicator dyes in the dual indicator reagent composition of the presentinvention. TABLE I includes indicator dyes that are presently used inassays for protein, plus several other suitable indicator dyes thatundergo a color transition in the pH range of approximately 2.8 toapproximately 5.2.

                  TABLE I                                                         ______________________________________                                        PROTEIN INDICATOR DYES                                                                                  Approximate pH of                                   Indicator Dye                                                                              Color Transition                                                                           Color Transition                                    ______________________________________                                        Bromochlorophenol                                                                          Yellow-Green 2.8                                                 Blue (BCPB)                                                                   Iodophenol Blue                                                                            Yellow-Blue  2.8                                                 (IPB)                                                                         Rose Bengal (RB)                                                                           Colorless-Pink                                                                             2.8                                                 Bromophenol Blue                                                                           Yellow-Blue  3.0                                                 (BPB)                                                                         Methyl Orange (MO)                                                                         Red-Yellow   3.0                                                 Tetrabromophenol                                                                           Yellow-Blue  3.3                                                 Blue (TBPB)                                                                   Bromopyrogallol                                                                            Yellow-Red   3.5                                                 Red (BPGR)                                                                    Bromocresol Green                                                                          Yellow-Green 4.3                                                 (BCG)                                                                         Tetrabromophenol-                                                                          Yellow-Green 4.3                                                 phthalein ethyl                                                               ester (TBEE)                                                                  Bromophenol Red                                                                            Yellow-Red   4.7                                                 (BPR)                                                                         HLO 301*     Colorless-Green                                                                            4.7                                                 Bromocresol Purple                                                                         Yellow-Purple                                                                              5.2                                                 (BCP)                                                                         ______________________________________                                         *HLO 301 is a tetracyclic dye having the chemical name                        8amino-11-aza-6-thia-[5,12-naphthacene-quinone].                         

The list of protein indicator dyes in TABLE I is a partial listincluding dyes that undergo a color transition at an acidic pH. Ingeneral, assays for protein have been conducted at an acidic pH andusing an indicator dye undergoing a color transition at an acidic pHbecause the indicator dye can interact more strongly with the protein atlow, acidic pH values. The increased interaction between the indicatordye and the protein at low pH values occurs because of a strongattraction between the positively-charged cationic protein molecule andthe negatively-charged anionic indicator dye molecule, and,additionally, because the acidic conditions serve to partially denatureproteins and therefore increase the ability of the protein to interactwith the indicator dye. However, it should be understood that otherindicator dyes, capable of interacting with proteins and undergoing acolor transition at a pH value above approximately 5.2, also can beemployed in the method of the present invention.

Accordingly, other indicator dyes, capable of undergoing a colortransition either in the acidic or in the neutral to alkaline pH range,also can be combined to yield a dual indicator reagent composition toafford improved color resolution and differentiation and increased assaysensitivity. However, each indicator dye included in the dual indicatorreagent composition must be capable of interacting with proteins, thetwo dyes must undergo color transitions within approximately the same pHrange and the dyes must undergo sufficiently different colortransitions. Examples of other pH indicator dyes that can be used in themethod of the present invention, and having a pH of color transitionranging from as low as 0.15 to as high as 14 are found in The MerckIndex, Ninth Edition, pages MISC-94 and MISC-95 (1976) and in Handbookof Chemistry and Physics, 51st Edition, pages D-106 through D-109(1970-1971). In addition, several other suitable pH indicator dyes areavailable commercially from numerous manufacturers and distributors.

In accordance with an important feature of the present invention,several suitable combinations of indicators are envisioned from theindicators listed in TABLE I. For example, methyl orange (MO) can becombined with bromochlorophenol blue (BCPB), bromophenol blue (BPB),tetrabromophenol blue (TBPB), or iodophenol blue (IPB) to produce acolor transition that provides enhanced color resolution, and thereforeincreased assay sensitivity. In each case, the intense red color ofmethyl orange (MO) will predominate prior to interaction with a protein;whereas after protein interaction and dye color transition, theresulting yellow color of methyl orange (MO) will be overcome by themore intense green or blue of the second dye. In addition, each of thesesecond indicator dyes (BCPB, BPB, TBPB and IPB) and methyl orange (MO)is capable of interacting with proteins, and each second indicator dyehas an approximate pH of color transition equal to, or approximatelyequal to, the pH of color transition for methyl orange (MO).Accordingly, it should be noted that rose bengal (RB) may not besuitable indicator dye to be combined with methyl orange (MO) to yield adual indicator reagent composition. Although rose bengal (RB) caninteract with proteins and has a pH of color transition thatapproximates the pH of color transition for methyl orange (MO), thecolor transition of rose bengal (RB) from colorless to pink issufficiently similar to the color transition of methyl orange from redto yellow such that the benefits of increased color resolution, andtherefore improved assay sensitivity, may not be achieved.

In another example, bromophenol red (BPR), having a yellow to red colortransition, can be combined with bromocresol green (BCG), having ayellow to green color transition, to give a color spectrum of yellow togreen to purple, in response to an increasing protein content of thetest sample. Similarly, bromophenol red (BPR), having a color transitionof yellow to red, can be combined with8-amino-11-aza-6-thia-[5,12-naphthacenequinone] (HLO 301), having acolor transition of colorless to green, to give a color spectrum ofyellow to orange to violet, in response to an increasing protein contentof the test sample.

To demonstrate the new and unexpected results achieved by the method ofthe present invention, a dual indicator reagent composition, includingthe indicators bromophenol blue (BPB) and methyl orange (MO), wasprepared, then used in an aqueous, liquid phase assay for total proteincontent of a test sample. Both bromophenol blue (BPB) and methyl orange(MO) interact with proteins and undergo a color transition atapproximately the identical pH of 3. The bromophenol blue (BPB) changescolor from yellow to a deep blue and the methyl orange (MO) changescolor from a deep red to yellow. A dual indicator reagent compositionincluding the appropriate amounts of bromophenol blue (BPB) and methylorange (MO), along with a suitable buffer produced the color transitionssummarized in TABLE II upon contact with standardized protein solutions.

                  TABLE II                                                        ______________________________________                                        COLOR TRANSITION OF METHYL ORANGE-BROMO-                                      PHENOL BLUE DUAL INDICATOR REAGENT COMPOSI-                                   TION UPON INTERACTION WITH STANDARDIZED                                       PROTEIN SOLUTIONS (pH = 3.2)                                                  Concentration of Standardized                                                 Protein Solution (mg/dL)                                                                            Observed Color                                          ______________________________________                                               0    (blank)       red or orange                                              10   (trace)       yellow or very                                                                light green                                                20                 light green                                                30                 green                                                      60                 blue green                                                 100                blue                                                       300                dark blue                                           ______________________________________                                    

In accordance with an important feature of the present invention, theimproved color resolution achieved by using the methylorange-bromophenol blue dual indicator reagent composition permitsdetection and differentiation between protein concentrations of 0, 10,20 and 30 mg/dL. In contrast, all prior art methods employing a singleindicator dye are unable to differentiate between protein levels in the0 to about 15 mg/dL range and provide only minimal differentiationbetween protein levels ranging from 0 to about 30 mg/dL. However, inaccordance with the present invention, increased assay sensitivity isachieved, especially at test sample protein levels of about 30 mg/dL andbelow to ultimately yield more accurate and meaningful assay results.

To perform an aqueous, liquid phase assay for total protein content, thedual indicator reagent composition is produced first. For example, adual indicator reagent composition is produced by dissolving 0.60 g(0.90 millimole) of bromophenol blue (BPB) and 0.60 g (1.83 millimole)of methyl orange (MO) in a sufficient amount of a 100 mM citrate bufferto yield one liter of an aqueous dual indicator reagent composition thatis 0.9 mM in bromophenol blue (BPB) and 1.83 mM in methyl orange (MO)buffered at pH 3.2. The presence and concentration of protein in a urinesample then was determined by adding one drop (approximately 50μL(microliters)) of urine to one Ml of the dual indicator reagentcomposition The color of the resulting aqueous solution changed from redto blue, therefore revealing the presence of approximately 100 mg/dL ofprotein in the urine sample.

In general, in the aqueous, liquid phase assay for protein, the dualindicator reagent composition is present in a sufficient amount to allowthe visual and/or instrumental detection and measurement of a colortransition. However, an excess amount of dual indicator reagentcomposition should be avoided such that any non-specific interactionswith non-protein test sample components are essentially precluded.Usually, a total concentration of dyes in the dual indicator reagentcomposition in the range of about 0.5 mM to about 5 mM is sufficient toprovide a detectable and differentiable color transition, eithervisually and/or by instrument, and to eliminate or minimize assayinterference through dye interaction with non-protein test samplecomponents. To achieve the full advantage of the present invention, ithas been found that a total dye concentration in the dual indicatorreagent composition in the range of from about 0.5 mM to about 2 mM isespecially preferred. Furthermore, it also has been found that inaddition to the citrate buffer used in the above example, the desired pHcan be maintained at an essentially constant level by using any suitablebuffer, such as malonate, lactate, trichloroacetate, sulfosalicylate,tartarate, phosphates, borates, acetates, piperazine-N,N'-bis(2-hydroxypropane)sulfonic acid (POPSO),N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES),3-N-(tris-hydroxymethyl)methylamine-2-hydroxypropanesulfonic acid(TAPSO), 2-([tris-(hydroxymethyl)methyl]amino)ethanesulfonic acid (TES),or other suitable buffers as are well known in the art.

Additionally, the two indicator dyes included in the dual indicatorreagent composition do not necessarily have to be present in equalamounts. The relative amount of each dye depends upon a variety ofparameters, including the intensity of the dye color transition andability of the dye to interact with proteins. However, it has been foundthat a ratio of the first indicator dye to the second indicator dyewithin a range of from about 5 to 1 to about 1 to 5, and preferably inthe range of from about 3 to 1 to about 1 to 3, provides the fulladvantages and benefits of the present invention.

Furthermore, in accordance with another important feature of the presentinvention, it is well within the experimental techniques of thoseskilled in the art of preparing test devices to design a system for theaqueous semiquantitative assay of proteins in urine and other liquidsamples by varying the relative amounts of aqueous solvent, dualindicator reagent composition, and urine sample, and by varying theidentity and amount of dyes and buffer, to provide detectable anddifferentiable color transitions, such that a comparison, eithervisually and/or by instrument, to color standards derived from solutionsof known protein concentration is possible.

In addition to the wet phase, aqueous assay for proteins, the dualindicator reagent composition can be used in dry phase, test pad assaysfor protein. The dry phase, test pad assay for protein that utilizes thedual indicator reagent composition is performed in accordance withmethods well known in the art. In general, the assay for protein isperformed by contacting the urine or other test sample with an analytedetection device that includes the dual indicator reagent composition.The analyte detection device can be dipped into the test sample, or thetest sample can be applied to the analyte detection device dropwise. Theresulting change in color of the analyte detection device demonstratesthe presence of protein; and, if so designed, the resulting colortransition can be compared to a standardized color chart to provide asemiquantitative measurement of the concentration of protein in theurine or test sample.

Typically, the analyte detection device is a reagent impregnated teststrip, designed either as a single pad test strip (to assay only for asingle analyte) or as a multiple pad test strip (to assay for severalanalytes simultaneously). For either type of reagent impregnated teststrip, the test strip includes a support strip, or handle, normallyconstructed from a hydrophobic plastic, and a reagent test pad,comprising a bibulous or nonbibulous carrier matrix. In general, thecarrier matrix is an absorbent material that allows the test sample tomove, in response to capillary forces, through the matrix to contact theindicator reagent composition and produce a detectable and measurablecolor transition.

The carrier matrix can be any substance capable of incorporating thechemical reagents required to perform the assay of interest, as long asthe carrier matrix is substantially inert with respect to the chemicalreagents, and is porous and/or absorbent relative to the liquid testsample. The expression "carrier matrix" refers to either bibulous ornonbibulous matrices that are insoluble in water and other physiologicalfluids and maintain their structural integrity when exposed to water andother physiological fluids. Suitable bibulous matrices include filterpaper, sponge materials, cellulose, wood, woven and nonwoven fabrics andthe like. Nonbibulous matrices include glass fiber, polymeric films, andpreformed or microporous membranes. Other suitable carrier matricesinclude hydrophilic inorganic powders, such as silica gel, alumina,diatomaceous earth and the like; argillaceous substances; cloth;hydrophilic natural polymeric materials, particularly cellulosicmaterial, like cellulosic beads, and especially fiber-containing paperssuch as filter paper or chromatographic paper; synthetic or modifiednaturally-occuring polymers, such as cellulose acetate, polyvinylchloride, polyacrylamide, polyacrylates, polyurethanes, crosslinkeddextran, agarose, and other such crosslinked and noncrosslinkedwater-insoluble hydrophilic polymers. Hydrophobic and non-absorptivesubstances are not suitable for use as the carrier matrix of the presentinvention. The carrier matrix can be of different chemical compositionsor a mixture of chemical compositions. The matrix also can vary inregards to smoothness and roughness combined with hardness and softness.However, in every instance, the carrier matrix must include ahydrophilic or absorptive material. The handle usually is formed fromhydrophobic materials such as cellulose acetate, polyethylene,terephthalate, polycarbonate or polystyrene, and the carrier matrix ismost advantageously constructed from bibulous filter paper ornonbibulous polymeric films.

To achieve the full advantage of the present invention, the dualindicator reagent composition is impregnated into a suitable carriermatrix and utilize in a dry phase test strip for the assay of protein ina test sample. The method of the present invention affords aneconomical, accurate and reliable assay for the total concentration ofprotein in test samples that can be performed at home or in thelaboratory. In addition, the method of the present invention allowsdetection, differentiation and measurement of low protein concentrationsin the test sample therefore making the assay more useful clinically.

In accordance with the method of the present invention, to perform a dryphase, test strip assay for protein, a aqueous solution, including fromabout 0.5 mM to about 5 mM total concentration of the two indicator dyesmethyl orange (MO) and bromophenol blue (BPB) first is prepared. Abibulous matrix, such as filter paper, then is saturated and impregnatedwith the aqueous solution containing the two dyes either by spreading,by immersing or by spraying the aqueous solution onto sheets or precutstrips of the filter paper. After removing the aqueous solvent by ovendrying in an air oven at about 50° C. for about 20 to 30 minutes, thefilter paper then is saturated and impregnated with a 250 mM citratebuffer at pH 3.2 either by immersion or by spraying. After oven dryingat about 50° C. for approximately 20 to 30 minutes, the filter paperimpregnated with the dual indicator reagent composition is cut to anappropriate size, such as a pad having dimensions from about 0.25 cm byabout 0.5 cm to about 0.5 cm by about 1.0 cm. Alternatively, it issometimes possible to combine all the ingredients into one impregnatingsolution and therefore avoid the necessity of a two-dip impregnationprocedure. The single dip procedure is especially recommended if the twodyes are sufficiently water soluble such that a second dip into thebuffer solution could cause some of the dyes to leach out of thebibulous matrix.

The filter paper impregnated with the dual indicator reagent compositionthen is secured to an opaque or transparent hydrophobic plastic handlewith double sided adhesive tape. The resulting test strip then wasdipped into a fresh, uncentrifuged urine sample for a sufficient time tosaturate the test pad with the sample. After waiting a predeterminedtime, such as 15 secs. to 60 secs., the test strip is examined, eithervisually or by instrument, for a response. The color transition, if any,of the test pad reveals the presence and/or concentration of protein inthe urine sample.

Analogous to the aqueous, liquid phase assay for protein describedabove, it is well within the experimental techniques of those skilled inthe art of preparing test devices to determine the proper balancebetween size of reagent pad, the strength of reagent impregnatingsolution, the amount of test sample, and the method of introducing thetest sample to the test strip, such as by pipetting rather than dipping,in order to design a semiquantitative assay for protein utilizing themethod and composition the present invention.

In many cases simple visual observation of the test strip provides thedesired information. If more accurate information is required, a colorchart bearing color spots corresponding to various known proteinconcentrations, can be prepared for the particular dual indicatorreagent composition used in the test strip. The resulting color of thetest strip after contact with the urine sample then can be compared withthe color spots on the chart to determine the protein concentration ofthe test sample.

If a still more accurate determination is required, a spectrophotometeror colorimeter can be used to more precisely determine the degree ofcolor transition. In addition, both the aqueous, liquid phase assay andthe dry phase, reagent strip assay can be made semiquantitative byemploying spectrophotometric or colorimetric techniques, as opposed tovisual techniques, in order to more reliably and more accurately measurethe degree of color transition, and therefore more accurately measurethe concentration of protein in the test sample, especially at lowerprotein concentrations, such as below 30 mg/dL.

As will be discussed more fully hereinafter in the detailed descriptionof FIGS. 1 through 4, the ability to detect, differentiate between andmeasure low concentrations of proteins in a test sample by employing adual indicator reagent composition surprisingly and unexpectedlyprovides a method of assaying for hard-to-detect low molecular weightproteins that may be present in the test sample. For example, thepresence of low molecular weight Bence Jones proteins in urine is adiagnostic indication that the patient suffers from leukemia or multiplemyeloma. However, according to present day methods, the detection ofBence Jones proteins in urine requires a heat and precipitationtechnique that is expensive and time-consuming. In addition, dry phasetest strips have not been used to assay for Bence Jones proteins becausethe high molecular weight proteins in urine, such as albumin, interferewith and mask the Bence Jones proteins assay. The high molecular weightproteins in urine are present in considerably greater quantities thanthe Bence Jones proteins and therefore the high molecular weightproteins preferentially react with the indicator dye. Furthermore,separation of the Bence Jones proteins from the other proteinconstituents in urine is as expensive and time-consuming as the presentday Bence Jones proteins assay, therefore making a protein separationstep, prior to a dry phase test strip assay, a useless manipulativetest. Accordingly, until the method of the present invention, no dryphase, test strip technique was available to accurately detect andmeasure the low concentrations of Bence Jones proteins usually found inurine.

Therefore, in accordance with an important feature of the presentinvention, it has been demonstrated that by impregnating the dualindicator reagent composition into a suitable carrier matrix, thepresence and concentration of Bence Jones proteins in a urine sample cane achieved by using a dry phase test strip. Surprisingly andunexpectedly, the dry phase test strip assay of Bence Jones proteins isaccomplished without separating the Bence Jones proteins from thesample, and without a masking of the Bence Jones proteins assay by themore abundant and interfering higher molecular weight proteins presentin the urine. As previously discussed, a dry phase test strip used forthe assay of proteins in test samples generally includes a carriermatrix comprising any absorbent matrix that is amenable to treatment andimpregnation with an indicator reagent composition; that permits theurine or other test sample to permeate the carrier matrix rapidly enoughto obtain protein assays relatively quickly; and that does notcontaminate the urine or other test sample either by test sampleextraction of components comprising the carrier matrix or by appreciablyaltering the urine or test sample in a way to make the subsequent assaysinconclusive, inaccurate or doubtful.

If the test strip is designed to assay for the total protein content ofa test sample, the carrier matrix can be any bibulous or nonbibulousmaterial that allows permeation by the test sample to saturate the assayarea of the test strip that is impregnated with the indicator reagentcomposition. To achieve the full advantage of the present invention, inthe assay for the total protein content of a test sample, the carriermatrix is a hydrophilic, bibulous matrix, including cellulosicmaterials, such as paper, and preferably filter paper. Filter paperpossesses all of the qualities required of a bibulous matrix of thepresent invention, plus the advantages of abundant supply, favorableeconomics, and a variety of suitable grades. Filter paper has been foundto be extremely satisfactory for use as a matrix material for suspendingand positioning both the indicator dyes and the buffers.

However, it has been found that in utilizing the dual indicator reagentcomposition in a method and device to determine the presence and/orconcentration of low molecular weight proteins, such as Bence Jonesproteins, in a test sample, the filter paper and cellulosic bibulousmatrices are unsuitable. The filter paper bibulous matrix and relatedbibulous matrices possess sufficient porosity to allow the relativelyhigh molecular weight proteins, such as albumin, to penetrate thebibulous matrix, and contact and interact with the impregnated dualindicator reagent composition to produce a color transition. Therefore,the proportionally large amount of relatively high molecular weightproteins present in the urine or other test sample precludes detectionof the proportionally small amount of low molecular weight proteinspresent in the test sample. As a result, by incorporating the dualindicator reagent composition into a carrier matrix possessing aporosity that is sufficiently small to exclude the relatively highmolecular weight proteins and simultaneously possessing a porosity thatis sufficiently large to allow penetration by the low molecular weightproteins, provides a method of detecting and/or differentiating betweenthe low levels of low molecular weight proteins in a test sample.

In accordance with an important feature of the present invention, it hasbeen found that a polymerized urethane-based film, layer or membraneprovides a carrier matrix having sufficient porosity to allowpenetration of the low molecular weight proteins, such as Bence Jonesproteins, and simultaneously to prevent penetration of the more abundantrelatively high molecular weight proteins, such as albumin. As will bedemonstrated in the embodiments of the present invention describedhereinafter, the dual indicator reagent composition can be incorporatedinto a polymerized urethane-based film, layer or membrane either afterforming the urethane-based film, layer or membrane or during theformation of the polymerized urethane-based film, layer or membrane.However, in either case, the polymerized urethane-based film, layer ormembrane must be treated with a suitable buffer, if required, before thefilm, layer or membrane can be used in a device to detect proteins.Furthermore, the polymerized urethane-based film, layer or membrane mustpossess a suitable porosity to permit the detection and measurement ofBence Jones proteins in test samples. It should also be understood thatpolymerized urethane-based films, layers, or membranes can be producedthat have a sufficiently high porosity to allow penetration by highermolecular weight proteins, like albumin, such that polymerizedurethane-based films, layers or membranes can be used with the dualindicator reagent composition of the present invention to assay a liquidsample for total protein content.

It has been found that in order to provide a polymerized urethane-basedfilm, layer or membrane of the appropriate porosity, a urethanecompound, such as a urethane prepolymer, is included in an incompletelycured form as a component of a polymerizable urethane-containingcomposition. The polymerizable urethane compound is dispersed ordissolved in a liquid vehicle. The liquid vehicle, being removable fromthe dispersion or solution during curing of the polymerizableurethane-containing composition, allows the polymerizableurethane-containing compound to dry and cure as a continuous layer, filmor membrane. The cured layer, film or membrane has the correct pore sizeto unexpectedly allow penetration of the relatively small, low molecularweight proteins and to exclude the relatively large high molecularweight proteins. The polymerized urethane-based film, layer or membraneis therefore suitable to function as the carrier matrix in a dry phasereagent test strip designed for the assay of Bence Jones proteins. Theurethane compound dispersed or dissolved in the continuous liquidvehicle phase can be oligomer, prepolymer, or incompletely curedpolymer. The polymerizable urethane-containing composition can be mixedwith the dual indicator reagent composition prior to curing, and thecarrier matrix, including the dual indicator reagent composition, thenis formed by curing the urethane-containing composition in layer form.The carrier matrix is cut into strips, then into pads, and secured to aplastic handle.

It has been found that the polymerizable urethane-containingcomposition, including a urethane compound like an oligomer, prepolymer,incompletely cured polymer or mixtures thereof that are capable ofpolymerization or further polymerization, form a cured film, layer ormembrane when cured or polymerized upon removal of the continuous liquidvehicle phase during curing to provide a film, layer or membraneunexpectedly having sufficient permeability to low molecular weightproteins and essentially no permeability to relatively large molecularweight proteins. The urethane compound, after dissolving or dispersingin a continuous phase, such as by including an emulsifier, can be curedin any known manner. Further, the solution or dispersion of the urethanecompound can include a suitable curing catalyst or can be heat cured solong as the solution or dispersion of the polymerizable urethanecompound is applied as a layer in the form of an incompletely curedsolution or dispersion. Generally, the urethane compounds that areuseful in accordance with the present invention are those that can bedissolved or dispersed in a liquid vehicle, such as an organic solvent,like dimethylformamide, and are polymerizable in the dissolved ordispersed form to yield an essentially colorless and continuous film,layer or membrane upon curing.

In accordance with one embodiment of the present invention, thepolymerizable urethane compound is a urethane prepolymer andparticularly a urethane prepolymer comprising essentially repeatingurethane units wherein the prepolymer chain is terminated at each endwith isocyanate functionalities. It has been found that the urethanecompound can be either neutral or cationic in character, or acombination of a neutral urethane compound and cationic urethanecompound can be used. Examples of suitable commercial urethaneprepolymers include DESMODERM KBH GRANULATE and DESMODERM KPKDISPERSION, both available commercially from BAYER AG.

The expression "urethane prepolymer" is understood to describe anessentially linear polymer of repeating urethane units. The urethaneprepolymer has at least two isocyanate functionalities per molecule, andthe polyurethane prepolymer should have a weight average molecularweight (M_(w)) of at least 50,000. Urethane prepolymers with weightaverage molecular weights below 50,000, for example down to about30,000, also are useful so long as the prepolymer form a continuousfilm, layer or membrane upon curing. The maximum M_(w) is one whereinthe urethane prepolymer can be solubilized or otherwise dispersed in aliquid vehicle or continuous phase, such as an organic solvent, likedimethylformamide. For the incompletely cured dispersed urethaneprepolymer weight average molecular weights of up to about 500,000 areexpected to be practical for the present invention. Upon curing, thereis no upper limit to the molecular weight of the film layer or membrane.It has been found that to exercise the full advantages of the presentinvention the M_(w) for the polymerizable urethane prepolymer is withinthe M_(w) range of about 70,000 to about 80,000.

The urethane compound, such as a urethane prepolymer, useful in themethod of the present invention can include other monomeric units thatare incorporated into the urethane compound by copolymerizing anisocyanate containing monomer, hydroxyl containing monomer and asuitable third monomeric unit into the urethane prepolymer. Similarly,the polyurethane compound useful in the method of the present inventioncan be either neutral (DESMODERM KBH), anionic or cationic (DESMODERMKPK) in nature. More particularly, DESMODERM KBH is a neutralthermoplastic granular polymerized urethane material, obtained byreacting 75 parts of a polyester of adipic acid, including 70 mol %ethylene glycol and 30 mol % 1,4-butanediol (M₂ =2,000); 25 parts of apolyester of adipic acid and 1,4-butanediol (M_(w) =2,250); 25 parts1,4-butanediol; and 85 parts diphenylmethanediisocyanate. Cationicurethanes in general are formed by a reaction of a polyisocyanate, apolyol and a hydroxyl-containing tertiary amine, wherein the amineportion of the poly- urethane is subsequently neutralized with anorganic acid, followed by dispersion of the neutralized polymerizedurethane in water. Accordingly, DESMODERM KPK is a cationic,emulsifier-free polymerized urethane dispersion of a reaction product of200 parts of a polyester of adipic acid, phthalic acid and ethyleneglycol (M_(w) =1,700); 50 parts toluenediisocyanate; 20 partsN-methyldiethanolamine; and 6 parts p-xylylene dichloride.

In any event, the urethane compound utilized in the present invention,after mixing with the other components of the urethane-containingcomposition, must cure to produce a film, layer or membrane that has aphysical and electrical charge structure that makes it permeable to lowmolecular weight proteins and impervious to relatively high molecularweight proteins. Furthermore, it should be understood that theurethane-containing composition can contain either a neutral urethanecompound, a cationic urethane compound or a mixture of a neutralurethane compound and a cationic urethane compound. The urethanecompound is present in the urethane-containing composition in a range offrom about 3% by weight to about 30% by weight, and preferably fromabout 5% by weight to about 20% by weight, based upon the total weightof the urethane-containing composition.

As will be discussed more fully hereinafter, the percentage of urethanecompound used in the urethane-containing composition, and the nature ofthe urethane compound, either neutral, cationic, or a neutral/cationicmixture, affects the degree of color resolution, the stability of colorproduction, and the speed of color production. Therefore, in accordancewith the method of the present invention, analyte test devices includinga urethane-based carrier matrix can be designed for improved colorresolution, increased color stability, or faster color production asrequired.

In addition to the polymerizable urethane compound, the polymerizableurethane-containing composition used to form the carrier matrix includesa dispersed inorganic phase, wherein the inorganic phase includes awater-insoluble inorganic compound, such as barium sulfate.

The urethane-containing composition includes from about 15% by weight toabout 40% by weight, and preferably from about 20% by weight to about30% by weight, based on the total weight of the urethane-containingcomposition, of a water-insoluble inorganic compound, such as bariumsulfate, as a filler. The exact identity of the inorganic compound usedas a filler is unimportant as long as the filler is white in color, soas not to interfere with color detection and measurement uponinteraction of the indicator dyes and the protein; and as long as theinorganic filler is essentially water-insoluble, such that dissolvedaniona and/or cations are not available to interfere chemically orphysically with the protein assay. Therefore, insoluble inorganiccompounds that can be used in accordance with the method of the presentinvention include calcium sulfate, titanium dioxide, alumina, zincoxide, magnesium oxide, calcium oxide, silicon dioxide, talc, magnesiumaluminum oxide, magnesium titanium oxide, barium oxide, barium sulfate,strontium sulfate and other similar, white, water-insoluble inorganiccompound, especially oxides; or mixtures thereof.

The insoluble inorganic compound is incorporated into theurethane-containing composition as a powder to help assure uniformdispersion of the insoluble inorganic compound throughout theurethane-containing composition. In addition, by utilizing an insolubleinorganic compound in powder form, the insoluble inorganic compound ismaintained uniformly dispersed throughout the urethane-containingcomposition during the curing process. The uniform dispersion of theinsoluble inorganic compound provides a polymerized urethane-based film,layer or membrane having the insoluble inorganic compound uniformlydispersed throughout the film, layer or membrane.

The polymerizable urethane-containing composition also can includeanionic surfactants to help wet the insoluble inorganic compound andtherefore assist in homogeneously dispersing the insoluble inorganiccompound throughout the urethane-containing composition. The anionicsurfactants can be present from 0% by weight up to approximately 5% byweight, based on the total weight of the urethane-containingcomposition. The anionic surfactant may further act to help stabilizethe color resulting from contact between protein and the dual indicatorreagent composition. The anionic surfactants found useful in the methodof the present invention are not necessarily limited to a particulartype, and include ammonium, alkylammonium, potassium and/or sodiumdodecylbenzene sulfonate, alkyl sulfonates, silylalkyl sulfonates, alkylsulfates, alkyl ethersulfates, dioctyl sulfosuccinate, alpha olefinsulfonates, and alkyl sarcosinates,; or mixtures thereof.

In addition, other surface active agents, such as silicon-containingmaterials, like a dimethylpolysiloxane fluid, can be incorporated intothe urethane-containing composition in weight percentages of up to 2%based upon the total weight of the urethane-containing composition.These silicon-containing materials possess a low surface tension andtherefore assist further in wetting the insoluble inorganic compound andalso act to alter the surface tension of the urethane-containingcomposition to provide a leveling affect to produce smooth and"polished" films, layers or membranes of uniform thickness.

As discussed previously, the urethane-containing composition alsoincludes a liquid vehicle, like an organic solvent, capable ofsolubilizing and/or dispersing the urethane compound and any anionicsurfactants or silicon-containing materials that may be present. Theliquid vehicle also must be capable of dispersing the insolubleinorganic salt. The organic solvent must be relatively inert such thatit will not react with the urethane compound and the solvent mustevaporate at relatively low temperatures to provide a dry urethane-basedfilm, layer or membrane. It has a been demonstrated that organic aproticsolvents, such as dimethylformamide, N-methyl pyrrolidone, and dimethylsulfoxide provide the required solvency to dissolve and disperse thecomponents of the urethane-containing composition, provide the requiredinertness to preclude reaction of the solvent with the urethanecompound, and possess the required vapor pressure to yield asolvent-free polymerized urethane-based film, layer or membrane. Theliquid vehicle, removed during curing, is included in theurethane-containing composition in an amount of at least 30%, andpreferably is present in an amount of at least 50% and up to about 90%by weight, based on the total weight of the polymerizableurethane-containing composition.

In accordance with one embodiment of the present invention,polymerizable urethane-containing compositions were mixed according tothe formulations outlined in Example 1. The urethane-containingcompositions A and B or Example 1, then were converted to urethane-basedfilms, layers or membranes according to an identical curing method.

EXAMPLE 1

    ______________________________________                                        Urethane-Containing Composition - A                                           DESMODERM KBH (Neutral                                                                              7.3%                                                    Urethane)                                                                     Sodium Dioctyl Sulfosuccinate                                                                       0.2%                                                    Barium Sulfate        22.0%                                                   Dimethylpolysiloxane Fluid                                                                          1.4%                                                    Sodium Dodecyl Benzenesulfonate                                                                     1.4%                                                    DESMODERM KPK (Cationic                                                                             10.0%                                                   Urethane)                                                                     Dimethylformamide     57.7%                                                   Total                 100.0%                                                  Urethane-Containing Composition - B                                           DESMODERM KBH (Neutral                                                                              5.8%                                                    Urethane)                                                                     Dralon U              1.6%                                                    Sodium Dodecyl Benzenesulfonate                                                                     0.3%                                                    Talc                  28.3%                                                   Dimethylpolysiloxane Fluid                                                                          0.1%                                                    Dimethylformamide     63.9%                                                   Total                 100.0%                                                  ______________________________________                                    

Dralon U is a sulfonated polymer of average molecular weight of 48,000and having the general structure illustrated in structural formula I.##STR1##

In the manufacture of both composition A and composition B of Example I,the components were thoroughly mixed using a high speed mixer until thecomposition was homogeneous. To cure either the composition A or B intoa film, layer or membrane, the composition is coated onto a transparent,impermeable plastic support. The thickness of the composition coating iscontrolled by using a doctor blade adjusted to a wet thickness of about150μ to about 750μ. Immediately after coating the plastic support withthe urethane-containing composition, the plastic support is immersedinto a circulating water bath maintained at a constant temperature ofabout 25° C. to about 43° C. The urethane-containing composition iscured in the water bath by immersing the composition-coated support inthe water bath for a time period ranging from 30 minutes to 16 hours.After curing, the film, layer or membrane can be air-dried oroven-dried. Reagents, such as dual indicator reagent composition, thenare impregnated into the dried film, layer or membrane as previouslydescribed. Alternatively, if the reagents comprising the dual indicatorreagent composition are soluble in the organic solvent used in themanufacture of the urethane-containing composition, likedimethylformamide, and if the reagents comprising the dual indicatorreagent composition are insoluble in water, the reagents can beincorporated into the urethane-containing composition and coated ontothe support the the urethane-containing composition prior to curing.

To show the new and unexpected results arising from using the dualindicator reagent composition to detect and measure the amount ofprotein in a test sample, and to further show the surprising resultsarising from incorporating the dual indicator reagent composition into aurethane-based film, layer or membrane, especially in regard to thedetection and measurement of low molecular weight proteins, like BenceJones proteins, in a test sample, color space plots were made for totalprotein assays and for Bence Jones protein assays obtained from dryphase test strips including a single indicator impregnated into a filterpaper bibulous matrix and into urethane-based carrier matrices and fromimpregnating a dual indicator reagent composition into a filter paperbibulous matrix and into polymerized urethane-based carrier matrices.

FIGS. 1-4 are color space plots obtained from contacting fourstandardized albumin solutions and from contacting a standardizedsolution of Bence Jones proteins with various dry phase test stripscomprising either a single indicator dye or a dual indicator reagentcomposition impregnated into a carrier matrix comprising either filterpaper or a polymerized urethane-based film, layer or membrane.

For example, FIG. 1 is the color space plot resulting from contacting adry phase test strip comprising the single indicator tetrabromophenolblue (TBPB) impregnated into a filter paper carrier matrix withstandardized solutions containing no albumin (0), 10 mg/dL albumin (10),50 mg/dL albumin (50), 100 mg/dL albumin (100) and 100 mg/dL Bence Jonesproteins (BJ). FIG. 2 is a color space plot for a dry phase test stripcomprising a dual indicator reagent composition includingtetrabromophenol blue (TBPB) and methyl orange (MO) impregnated into afilter paper carrier matrix that resulted from contacting the samestandardized solutions of albumin and Bence Jones proteins. Similarly,FIG. 3 is a color space plot obtained from contacting the standardizedprotein solutions with a dry phase test strip comprising the singleindicator tetrabromophenol blue (TBPB) incorporated into a polymerizedurethane-based film obtained by curing composition A of Example 1. FIG.4 is the color space plot obtained from contacting the standardizedprotein solutions with a dry phase test strip comprising a dualindicator reagent composition including tetrabromophenol blue (TBPB) andmethyl orange (MO) incorporated into a polymerized urethane-based filmobtained by curing composition B of Example 1.

As illustrated in FIGS. 1-4, a color space plot includes three axes, theL*, A* and B* axes. The values of L* plotted on the vertical axis are ameasure of the intensity of color, whereby a large L* value denotes alight color and L*=0 denotes a completely black color. The horizontal A*axis is a measure of the color transition from green to red, whereby themore positive the A* value, the more red the color, and analogously, themore negative the A* value, the more green the color. Similarly, thethird axis, B*, is a measure of the color transition from blue toyellow, whereby the greater the value of B*, the more yellow the color,and analogously the smaller the value of B*, the more blue the color.

The color space difference (ΔE) is calculated from the followingequation: ##EQU1## wherein: L₁ *, A₁ *, and B₁ * are the color spacevalues determined for a first standardized protein solution;

L₂ *, A₂ * and B₂ * are the color space values determined for a secondstandardized protein solution having a different protein concentrationfrom the first standardized protein solution; and

ΔE is the color space difference between the color space plots of thefirst and second standardized protein solutions.

The color space difference (ΔE) is the straight line distance betweentwo points in a three-dimensional color space plot. Theoretically, acolor space difference of 1 is the smallest color difference the humaneye can distinguish. However, because of the inherent differencesbetween the visual capabilities of individuals, a color space difference(ΔE) of about 5 is required in order to practically and confidentlydistinguish between colors.

The L*, A* and B* values plotted on the color space plots of FIGS. 1through 4 are calculated from the percent reflectance measurements takenat sixteen different wavelengths evenly spaced between 400 nm(nanometers) and 700 nm using standard equations well-known in the art.In general, the percent reflectance at each of the sixteen differentwavelengths is multiplied by the intensity of the light at thatwavelength. These values then are multiplied by standard weighingfunctions for the color red, green and blue, and finally added together.These calculations yield three tristimulus values X, Y and Z, and L*, A*and B* are calculated from the X, Y and Z tristimulus values using thefollowing equations:

    L*=116×[(Y/Yo)1/3-16)]                               (Eq. 2)

    A*=500×[(X/Xo)1/3-(Y/Yo)1/3]                         (Eq. 3)

    B*=200×[(Y/Yo)1/3-(Z/Zo)1/3]                         (Eq. 4)

wherein:

Xo, Yo and Zo are the tristimulus values for perfect white (i.e.reflectance =100% at all wavelengths), and X, Y and Z are the colorspace difference values calculated as described above from the sixteenwavelengths between 400 nm and 700 nm.

From the color space plots of FIGS. 1 through 4, the color spacedifferences (ΔE) were calculated, and summarized in TABLE III. Ininterpreting TABLE III, the term ΔE(Alb 10-0) is the color spacedifference between protein assays for protein solutions containing 10mg/dL of albumin and 0 mg/dL of albumin. Similarly, the term ΔE(Alb50-0)is the color space difference between protein assays for proteinsolutions containing 50 mg/dL of protein and 0 mg/dL of protein. Theterms ΔE (Alb100-0) and ΔE (BJ100-0) are analogously defined.

                                      TABLE III                                   __________________________________________________________________________    COLOR SPACE DIFFERENCES (ΔE) FOR SINGLE AND DUAL                        INDICATOR REAGENT SYSTEMS IN FILTER PAPER AND                                 POLYMERIZED URETHANE-CONTAINING MATRICES                                      FIG.                                                                             CARRIER           ΔE                                                                           ΔE                                                                           ΔE                                                                            ΔE                                 NO.                                                                              MATRIX  INDICATOR(S)                                                                            (Alb10-0)                                                                          (Alb50-0)                                                                          (Alb100-0)                                                                          (BJ100-0)                                __________________________________________________________________________    1  Filter  Tetrabromophenol                                                                        4.8  19.2 25.5  4.4                                         Paper   Blue                                                               2  Filter  Tetrabromophenol                                                                        9.1  22.0 30.2  12.2                                        Paper   Blue and Methyl                                                               Orange                                                             3  Urethane                                                                              Tetrabromophenol                                                                        3.2  9.9  20.6  29.3                                        Composition A                                                                         Blue                                                               4  Urethane                                                                              Tetrabromophenol                                                                        7.1  21.6 30.4  48.9                                        Composition B                                                                         Blue and Methyl                                                               Orange                                                             __________________________________________________________________________     0 = Albumin 0 mg/dL and Bence Jones protein 0 mg/dL                           Alb10 = Albumin 10 mg/dL                                                      Alb50 = Albumin 50 mg/dL                                                      Alb100 = Albumin 100 mg/dL                                                    BJ100 = Bence Jones proteins 100 mg/dL                                   

As illustrated in the color space plot of FIG. 1 and in TABLE III,protein assays were conducted on standardized solutions includingalbumin and Bence Jones proteins using a dry phase test strip havingonly a single indicator, tetrabromophenol blue, impregnated into afilter paper carrier matrix. From FIG. 1 and TABLE III, it is found thatthe color space difference between a solution containing 10 mg/dL ofalbumin and a solution containing no albumin is 4.8. Because the humaneye can normally differentiate only between colors having a color spacedifference of approximately 5, this assay would be inconclusive as towhether the sample contained any albumin because the colordifferentiation between the test strip contacting the 0 mg/dL albuminsolution and the test strip contacting the 10 mg/dL test strip could notbe determined. At best, the assayer could estimate that the samplecontained from 0 mg/dL albumin to about 10 mg/dL albumin.

Similarly, FIG. 1 and TABLE III demonstrate that an assayer could notdetermine the concentration of Bence Jones proteins in a test samplecontaining from 0 mg/dL of Bence Jones proteins to about 100 mg/dL ofBence Jones proteins because the color space difference provided by ananalyte device having a single dye impregnated into a filter papermatrix is only 4.4, or a color space difference that is barelydetectable by a normal human eye. TABLE III and FIG. 1 further shownthat the human eye can detect color differences resulting from thepresence of 50 mg/dL and 100 mg/dL of albumin because the color spacedifference are 19.2 and 25.5, respectively.

However, surprisingly and unexpectedly, by impregnating a filter papermatrix with a dual indicator reagent composition of the presentinvention, an assayer can visually differentiate between samplescontaining 0 mg/dL of albumin and 10 mg/dL albumin. From FIG. 2 andTABLE III, a color space difference (ΔE) between a solution containing10 mg/dL of albumin and a solution containing no albumin is 9.1 whenusing a dual indicator reagent composition including tetrabromophenolblue and methyl orange. Such a color space difference is sufficient tobe discernible by the human eye, and shows a substantial improvementover the color space difference of 4.8 afforded by the single indicatordye of FIG. 1. Similarly, an assayer can visually detect Bence Jonesproteins in a test sample because the color space difference between a a100 mg/dL solution of Bence Jones proteins and a 0 mg/dL solution ofBence Jones proteins is 12.2. Such a degree of color difference issufficient to allow color differentiation by the human eye. Similarly,TABLE III and FIG. 2 shows enhanced color differentiation for the 50mg/dL and 100 mg/dL albumin solutions compared to the solutioncontaining no albumin.

In regard to FIG. 3, it is demonstrated that a single indicator dyeimpregnated into a polymerized urethane-based film, layer or matrix doesnot provide a method to determine the presence and/or concentration oflow levels of albumin in a test sample. For a solution containing 10mg/dL of albumin, the color space difference (ΔE) compared to a controlsolution containing 0 mg/dL albumin was only 3.2. This color spacedifference is insufficient for differentiation by the human eye.However, it is surprising that the polymerized urethane-based filmmatrix provided dramatically increased sensitivity in regard to BenceJones proteins as the color space difference in FIG. 3 rose to 29.3compared to the ΔE in FIG. 1 of 4.4 wherein a filter paper matrix wasused.

Unexpectedly, even greater sensitivity in regard to Bence Jones proteinsassay was found in FIG. 4, wherein a dual indicator reagent compositionwas incorporated into a polymerized urethane-based film matrix. Comparedto FIG. 3, the color space index increased from 29.3 to 48.9 showing anunexpected increase in color resolution and sensitivity towards BenceJones proteins. FIG. 4 further shows the benefits of using a dualindicator reagent composition incorporated into a polymerizedurethane-based film matrix to assay for albumin because the ΔE valueincreased to the visually perceptible level of 7.1 for a solutioncontaining 10 mg/dL of albumin compared to the visually imperceptible ΔEvalue of 3.2 from FIG. 3, wherein a single indicator dye was utilized.

Overall, FIGS, 1-4 and TABLE III shows that a dual indicator reagentcomposition impregnated into a filter paper matrix or into a polymerizedurethane-based film matrix improves color resolution and assaysensitivity in the assay for the total protein content of a liquid testsample, especially at low protein levels of less than 30 mg/dL. Themethod and composition of the present invention allow visualdifferentiation of color transitions resulting from contact of thereagent-containing carrier matrix with a test sample containing proteinat levels of between 0 mg/dL and 10 mg/dL, thereby providing moreaccurate and trustworthy assays. The present invention further providesa method to quickly and accurately test for Bence Jones proteins, andother low molecular weight proteins, in a test sample by providing acarrier matrix that essentially removes interfering high molecularproteins and by providing a reagent composition of sufficientsensitivity to allow detection and measurement of low concentrations oflow molecular weight proteins.

It has been demonstrated that color differences are improved by usingthe dual indicator reagent composition, regardless of whether thecarrier matrix is filter paper or a polymerized urethane-based film,membrane or layer. In addition, employing the dual indicator reagentcomposition in a polymerized urethane-based film matrix showsdramatically increased sensitivity to low molecular weight proteinstherefore providing a simple dry phase test strip procedure to assay forlow molecular weight proteins. As demonstrated in FIGS. 1-4 and in TABLEIII, assaying a solution containing 100 mg/dL of Bence Jones proteinswith a single indicator dye incorporated into a filter paper matrix gavean imperceptible color difference of 4.4 compared to assaying a solutioncontaining on Bence Jones proteins. However, color resolution and assaysensitivity is improved by incorporating the same single dye into apolymerized urethane-containing matrix such that the color difference isan easily perceptible 29.3. Furthermore, using the dual indicatorreagent composition incorporated into a polymerized urethane-based filmmatrix further dramatically improves the color resolution and assaysensitivity such that the color difference increases to an unexpectedlevel of 48.9.

In regard to using a polymerized urethane-based film, layer or membraneas the carrier matrix for a dual indicator reagent composition in theassay for low molecular weight proteins, it has been found that not allurethane-based membranes respond identically to contact withprotein-containing solutions, and therefore, several urethane-based filmmatrices are unsuitable because of high blank color development,insufficient color differentiation between protein levels and/or colorleaching into aqueous phase. It has been shown that the two polymerizedurethane-based film matrices obtained by curing composition A orcomposition B of Example 1 do not demonstrate these disadvantages andtherefore are preferred. It should be emphasized however thatcompositions A and B are not the only compositions that can be utilizedaccording to the method of the present invention as matrices to providegood protein determinations.

Nevertheless, a membrane, layer or film obtained by curing eithercomposition A or by curing composition B of Example 1 has advantages anddisadvantages. For example, a membrane of film obtained by curingcomposition A of Example 1 gives excellent color differentiation andexcellent color stability even after the test sample is wiped dry fromthe membrane. For example, for analyte test devices using membranes offilms derived from curing composition A of Example 1, the colortransition resulting from contact with albumin or Bence Jones proteinsshowed no visual deterioration in color intensity or depth over aseveral day period. However, color formation in films derived fromcomposition A is slow, and therefore this film may have limitations ifused in the usual dip-and-read manner. As a result, when using the filmmatrix derived from composition A, the test sample is pipetted onto thefilm matrix and allowed to contact the film matrix for approximately 2minutes. The color generated in response to the albumin contact then isdetermined either visually or instrumentally and either with the testsample remaining in contact with the matrix or after the sample is wipedfrom the matrix.

A urethane-based film matrix obtained by curing composition B of Example1 also offers very good color resolution and differentiation. However,unlike a carrier matrix formed by curing composition A or Example 1,color formation on a film matrix obtained by curing composition B ofExample 1 is fast, and therefore, this film matrix can be used in theusual dip-and-read format in the assay for albumin. However, in theassay for Bence-Jones proteins color development is slow, in that 2minutes is required for full color development. Therefore, the teststrip would have to remain dipped in the urine sample for a relativelylong time to generate a color transition. This disadvantage is overcomeby pipetting the urine sample onto a test pad and allowing a 2 minuteresponse time before examining the test strip for a response.Furthermore, after the sample is wiped off the matrix, the colorgenerated in response to the protein content begins to fade, andtherefore the degree and depth of color transition must be determinedimmediately after removing the liquid test sample from the test strip.

Therefore, in accordance with an important feature of the presentinvention, more accurate and reliable assays for total protein content,or for low molecular weight protein content, in urine and other liquidtest samples can be performed by utilizing a dual indicator reagentcomposition. The dual indicator reagent composition improves the colorresolution of the assay and therefore improves assay sensitivity,especially at low albumin levels of approximately 30 mg/dL and below.Furthermore, by performing the assay with a dry phase test strip thatincludes a polymerized urethane-based membrane, film or layer as thecarrier matrix for the dual indicator reagent composition, a new andunexpectedly accurate method of determining the presence and/orconcentration of low molecular weight proteins, like Bence Jonesproteins, in the test sample is provided.

Obviously, many modifications and variations of the invention ashereinbefore set forth can be made without departing from the spirit andscope thereof and therefore only such limitations should be imposed asare indicated by the appended claims.

I claim:
 1. A method of manufacturing a test article for determining thepresence of a protein in a test fluid comprising:mixing a predeterminedquantity of a reagent composition into an incompletely curedpolymerizable urethane material dispersed in a removable liquid vehicleto form a reagent-containing matrix material; forming saidreagent-containing matrix material into a layer; and drying said layerand polymerizing said polymerizable urethane material while removingsaid liquid vehicle to form a dried polyurethane matrix material layerpermeable to said protein and containing a reagent composition capableof reaction with said protein when said predetermined protein penetratesinto said dried polyurethane matrix material layer.
 2. The method ofclaim 1 wherein the matrix is formed from a layer of a compositioncomprising a dispersed, polymerizable urethane compound in the removableliquid vehicle, and a portion of said liquid being removed duringpolymerization of said urethane compound in dispersed, layered form, anddried to form said matrix having said reagent composition homogeneouslyimmobilized therein in a predetermined concentration.
 3. The method ofclaim 2 wherein the matrix if formed from a layer of a compositionfurther comprising a water-insoluble inorganic compound.
 4. The methodof claim 1 wherein the reagent composition comprises:a first indicatordye capable of undergoing a detectable and measurable color transitionfrom a first color to a second color; a second indicator dye capable ofundergoing a detectable and measurable color transition at approximatelythe same pH as the first indicator dye to a color that differs from thesecond color of the first indicator dye; and a suitable buffer tomaintain a constant pH sufficiently close to the color transition pH ofthe first indicator dye and the color transition pH of the secondindicator dye; wherein each dye is capable of preferentially interactingwith protein in a liquid sample to undergo color transition of the dyesin a manner that the color transitions do not mutually interfere witheach other and each of the dyes has approximately the same affinity forprotein in the liquid sample.
 5. A method of manufacturing a test deviceincluding a continuous layer of urethane based polymer permeable toBence Jones proteins while screening out albumin and other highmolecular weight proteins found in biological fluidscomprising:dispersing a polymerizable urethane compound in a removableliquid vehicle to form an incompletely cured polymerizable layer formingcomposition; applying a layer of said incompletely cured compositiononto a support surface; and polymerizing said urethane compound while inlayer form and removing a substantial portion of the liquid vehicleduring polymerization of said urethane compound in dispersed, layerform, to form a cured, continuous polymerized urethane based polymerpermeable to said Bence Jones proteins; wherein a reagent compositioncapable of interaction with Bence Jones proteins to produce a detectablecolor change is added to the polymerizable layer forming compositionbefore polymerizing said polymerizable urethane compound.
 6. The methodof claim 5 wherein said support surface comprises a continuous sheet ofpolyethylene terephthalate, cellulose acetate, polystyrene orpolycarbonate.